Systematic investigation and understanding of cells depends on the tools available with which to probe cell function. Although cells exhibit complex intracellular and morphological behavior, and carry out functions over time, limitations in the ability to genetically probe these processes directly hinders the ability to understand cell function. Currently, an ability to observe cells microscopically and then arbitrarily sort subpopulations of the observed cells, on a reasonably large scale, is lacking.
Flow cytometry and the related fluorescence-activated cell sorting (FACS) flow cells in a buffer single-file past an interrogation point, allowing high-throughput (>10,000 cells/sec) analysis of light scatter and whole-cell fluorescence, however, flow sorters do not image, making them unable to sort based upon morphological or intracellular information, and their very nature as flowing systems makes it difficult to observe the same cell at widely spaced timepoints, as would be needed to screen for temporal behavior. Attempts have been made to create flow cytometers that can obtain intracellular and dynamic information, such as slit-scan flow cytometers and imaging flow cytometry that can image micron-sized particles as they pass the observation point, however they cannot sort cells, and cannot assay cells adhered to substrates as is desirable for imaging and for investigating processes specific to adherent cells.
The complementary technology to flow cytometry is microscopy. In microscopy, cells are randomly arrayed on a coverslip or multi-well plate and then observed. Observation can include intracellular and/or temporal imaging. Many advanced microscopy technologies have been developed over the years to enhance screening capabilities. Today, all major microscopy manufacturers offer fluorescence microscopes with automated stages, focusing, objectives, fluorescence filters, etc. In conjunction with commercially available software (e.g., Metamorph by Universal Imaging), these allow computer-controlled location and observation of cells over time and space. In addition, specific high-throughput imaging systems have been optimized for pharmaceutical screening (e.g., Cellomics, Automated Cell Inc.). All conventional microscopy technologies, however, are limited in their ability to viably isolate cells following imaging.
Cell-based genetic screens, employing the observation of cells and their isolation when exhibiting a desired phenotype, has been accomplished via the use of microscopy, however, its use is severely limited in its ability to isolate positive-responding cells. The premiere isolation technique, fluorescence-activated cell sorting (FACS), another means of isolating cells with a desired phenotype is limited in terms of the phenotypic changes that can be observed. A gap therefore exists between what is observable micropcopically, and what may be isolated, with a clear and present need for a technology that effectively bridges the gap.